EP0952846A1 - Prolactin as a vaccine adjuvant - Google Patents

Prolactin as a vaccine adjuvant

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Publication number
EP0952846A1
EP0952846A1 EP95910999A EP95910999A EP0952846A1 EP 0952846 A1 EP0952846 A1 EP 0952846A1 EP 95910999 A EP95910999 A EP 95910999A EP 95910999 A EP95910999 A EP 95910999A EP 0952846 A1 EP0952846 A1 EP 0952846A1
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EP
European Patent Office
Prior art keywords
prolactin
composition
vaccine
human
cdna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95910999A
Other languages
German (de)
French (fr)
Other versions
EP0952846A4 (en
Inventor
Susan Richards
Johanne Kaplan
Richard Moscicki
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genzyme Corp
Original Assignee
Genzyme Corp
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Publication date
Application filed by Genzyme Corp filed Critical Genzyme Corp
Publication of EP0952846A1 publication Critical patent/EP0952846A1/en
Publication of EP0952846A4 publication Critical patent/EP0952846A4/xx
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57554—Prolactin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the antigens used in the vaccine are not sufficiently immunogenic to raise the antibody titer to sufficient levels to provide protection against subsequent challenge or to maintain the potential for mounting these levels over extended time periods.
  • the vaccine may be deficient in inducing cell-mediated immunity which is a primary immune defense against bacterial and viral infection.
  • the vaccine In order to obtain a stronger humoral and/or cellular response, it is common to administer the vaccine in a formulation containing an adjuvant, a material which enhances the immune response of the patient to the vaccine.
  • adjuvants for vaccines are oil preparations and alum. The mechanisms by which such adjuvants function are not understood, and whether or not a particular adjuvant preparation will be sufficiently effective in a given instance is not predictable.
  • the present invention relates to a composition for enhancing the immune response of an animal to an infectious disease vaccine wherein the composition comprises prolactin.
  • the composition is human prolactin and the animal to be vaccinated is, as well, human.
  • the present invention further relates to a composition for enhancing the immune response of an animal to an infectious disease vaccine wherein the composition comprises prolactin cDNA.
  • the composition comprises prolactin cDNA.
  • Human prolactin cDNA is preferred.
  • the invention relates to a method of enhancing the immune response of a subject animal to an infectious disease vaccine comprising co-administering an effective amount of prolactin or prolactin cDNA along with a vaccine.
  • Figure 1 shows the amino acid sequence for the prolactin protein.
  • Figure 2 shows the nucleic acid sequence for the prolactin cDNA.
  • FIG. 3 is a graph illustrating the Bovine serum albumin (BSA)-specific antibody response of rats immunized with BSA alone or BSA + prolactin.
  • BSA Bovine serum albumin
  • Figure 4 is a graph illustrating a comparison of the BSA- specific proliferative response of rat PBL, at 101 day time point, between four rats receiving BSA alone versus BSA + prolactin.
  • prolactin refers to a polypeptide obtained from tissue cultures or by recombinant techniques and other techniques known to those of skill in the art, exhibiting the spectrum of activities characterizing this protein.
  • the word includes not only human prolactin (hPRL), but also other mammalian prolactin such as, e.g., mouse, rat, rabbit, primate, pig and bovine prolactin.
  • hPRL human prolactin
  • r-PRL The recombinant PRL (r-PRL) is preferred herein.
  • prolactin refers to prolactin having comparable biological activity to native prolactin prepared by recombinant DNA techniques known by those of skill in the art.
  • the gene coding for prolactin is excised from its native plasmid and inserted into a cloning vector to be cloned and then inserted into an expression vector, which is used to transform a host organism. The host organism expresses the foreign gene to produce prolactin under expression conditions.
  • the term "adjuvant” has its conventional meaning, i.e., the ability to enhance the immune response to a particular antigen. Such ability is manifested by a significant increase in immune-mediated protection.
  • the term “genetic adjuvant” refers to prolactin cDNA which comprises the complement to the DNA sequence encoding the prolactin protein as defined above. The sequence for prolactin cDNA is shown in Figure 2.
  • Formulations containing prolactin for adjuvant purposes are most conveniently administered by intramuscular or subcutaneous injections or intraperitoneal although other methods of administration are possible.
  • Standard formulations are either liquid injectables or solids which can be taken up in suitable liquids as suspensions or solutions for injection.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and so forth.
  • Nontoxic auxiliary substances, such as wetting agents, buffers, or emulsifiers may also be added.
  • Prolactin can be administered separately from the vaccine or in combination with the vaccine.
  • the composition administered contains an immunogen that is effective in eliciting a specific response to a given pathogen or antigen, a pharmaceutically acceptable vaccine carrier and an immunopotentiating amount of prolactin.
  • the vaccine will normally be administered per manufacturer's instructions.
  • Other adjuvants may be administered either with the vaccine or together with the prolactin.
  • Prolactin will typically be used to enhance the protection afforded by animal or human vaccines that are considered "weak” (i.e., provide diminished protection in terms of level, extent, and/or duration).
  • vaccines are bacterins such as Pseudomonas Staphylococcal, Enterotoxin Streptococci, cytomegalovirus, HIV, Bordetella bacterin, Escherichia coli bacterins, Haemophilus bacterins, Leptospirosis vaccines, Moraxella bovis bacterin, Pasteurella bacterin and Vibrio fetus bacterin and attenuated live or killed virus products such as bovine respiratory disease vaccine (infectious bovine rhinotracheitis, parainfluenza-3, respiratory syncytial virus), bovine virus diarrhea vaccine, equine influenza vaccine, feline leukemia vaccine, feline respiratory disease vaccine (rhinotracheitis-calicipneumonitis viruses), canine parvovirus vaccine, transmiss
  • prolactin can enhance the immune response to an immunogen and thereby function as a vaccine adjuvant
  • exogenous administration of the prolactin gene would result in the expression of prolactin in vivo which would be available to function as an adjuvant to any immunogen whether administered through conventional means or via gene inoculation.
  • the "genetic adjuvant” could be produced by inserting prolactin cDNA into a DNA delivery vehicle (e.g., plasmid vectors, liposomes, viral vectors). This could be accomplished as described by Pellegrini I., et al., Molec.
  • the injection sequence would be optimized per immunogen, i.e., the prolactin cDNA could be co-administered with the immunogen or immunogen cDNA, or administered in advance or subsequent to their administration. It is believed that the prolactin cDNA could be inserted into the same DNA delivery vehicle. Various routes of administration could be used.
  • Peripheral blood lymphocytes were isolated from the blood of normal human volunteers by density gradient centrifugation on Ficoll Paque (Pharmacia). Heparinized blood was diluted 3 fold in phosphate-buffered saline (PBS) and centrifuged at 2000 rpm for 20 minutes. The buffy coat, located on the surface of the red blood cell pellet and consisting of white blood cells, was collected and diluted with an equal volume of PBS. The diluted buffy coat was layered on Ficoll Paque (6 mis of buffy coat on 4 mis of Ficoll Paque in a 15 ml tube) and centrifuged for 30 minutes at 1400 rpm.
  • PBS phosphate-buffered saline
  • PBL The PBL layer, found at the Ficoll-plasma interface, was collected and the cells were washed three times in PBS. PBL were then resuspended at 2x10 6 /ml in serum-free AIM-V medium from Gibco and added to the wells of round bottom 96 well microtiter plates in a 100 ⁇ l volume (2x10 5 PBL/well).
  • T cell mitogen concanavalin A (Con A; 0.2 ⁇ g/ml) was added in a 50 ⁇ l volume together with 50 ⁇ l of varying concentrations of r-hPRL (0-1000 ng/ml final). Cultures were done in triplicate. The cells were incubated at 37 ° C/5% CO2 for 72 hours and the amount of proliferation measured by tritiated thymidine incorporation.
  • Tritiated thymidine (0.5 ⁇ Ci/well) was added for the last 18 hours of incubation and cell-associated radioactivity was measured by scintillation counting after harvesting the cells onto glass fiber filters using a Skatron 96 well cell harvester.
  • Results obtained with cells from different individuals, shown in Table 1 below, indicated that r-hPRL was able to enhance the proliferative response of T lymphocytes to a suboptimal concentration of Con A. This co-mitogenic activity was best observed with r-hPRL concentrations of 1 -10 ng/ml, illustrated in Figure 3.
  • r-hPRL To test the ability of r-hPRL to enhance the proliferative response of human T cells to a specific antigen, PBL were incubated with various concentrations of r-hPRL and streptokinase, a common antigen to which most individuals are exposed. Cultures were performed in triplicate in the wells of 96 well round bottom microtiter plates and consisted of 100 ⁇ l PBL (2x10 5 /well), 50 ⁇ l streptokinase (25 ⁇ g/ml final) and 50 ⁇ l of r-hPRL at varying concentrations (0-1000 ng/ml final). Proliferation was measured by tritiated thymidine incorporation after 6 days of culture at 37 ° C/5%C02-
  • Twenty-four 150 gram male Sprague-Dawley rats were divided into 4 groups.
  • the control group received an intraperitoneal injection of 10 ⁇ g BSA mixed with alum.
  • the other 3 groups received intraperitoneal injections of 10 ⁇ g BSA mixed with alum along with either 180 ⁇ g prolactin, 375 ⁇ g prolactin or 750 ⁇ g prolactin.
  • Tail vein bleeds were taken weekly for 4 weeks and the serum evaluated for antibody to BSA by a Radioimmunosorbent Assay (RIA).
  • RIA Radioimmunosorbent Assay
  • the animals were boosted after the 4th bleed with 10 ⁇ g BSA mixed with alum.
  • Tail vein bleeds were taken over a 7 week period to obtain serum which was evaluated for the development of antibody to BSA by RIA.
  • Bovine serum albumin BSA-specific proliferation of peripheral blood lymphocytes from rats immunized with BSA +/- r-hPRL
  • BSA Bovine serum albumin
  • PBL were then washed twice in PBS and resuspended at 5x10 6 /ml in RPMI-1640 medium supplemented with 100 u/ml penicillin, 100 ⁇ g/ml streptomycin, 20 mM Hepes buffer, 2 mM L-glutamine, 5x10" 5 M 2-mercaptoethanol and 5% heat-inactivated fetal calf serum.
  • PBL were added to the wells of flat bottom 96 well microtiter plates in a 100 ⁇ l volume (5x10 ⁇ cells/well) and cultured in the presence of medium alone (background control) or 1000 ⁇ g/ml BSA added in a 100 ⁇ l volume. Cultures were done in triplicate. Proliferation was measured by tritiated thymidine incorporation after 5 days of culture at 37 ° C/5% C ⁇ 2-

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Abstract

The present invention relates to a composition for enhancing the immune response of an animal to an infectious disease vaccine wherein the composition comprises prolactin. Preferably, the composition is human prolactin and the animal to be vaccinated is, as well, human. The present invention further relates to a composition for enhancing the immune response of an animal to an infectious disease vaccine wherein the composition comprises prolactin cDNA. Human prolactin cDNA is preferred.

Description

PROLACTIN AS A VACCINE ADJUVANT
Background of the Invention The use of vaccines to prevent diseases in humans, farm livestock, sports animals and household pets is a common practice, and considerable effort has been, and is being, made to extend this practice to cover a more extensive array of diseases to which these patients are subject. For example, the use of rabies vaccine in animals is by now commonplace, and efforts are being made to obtain suitable vaccines to immunize animals against other diseases.
One problem that frequently is encountered in the course of active immunization is that the antigens used in the vaccine are not sufficiently immunogenic to raise the antibody titer to sufficient levels to provide protection against subsequent challenge or to maintain the potential for mounting these levels over extended time periods. Another problem is that the vaccine may be deficient in inducing cell-mediated immunity which is a primary immune defense against bacterial and viral infection.
In order to obtain a stronger humoral and/or cellular response, it is common to administer the vaccine in a formulation containing an adjuvant, a material which enhances the immune response of the patient to the vaccine. The most commonly used adjuvants for vaccines are oil preparations and alum. The mechanisms by which such adjuvants function are not understood, and whether or not a particular adjuvant preparation will be sufficiently effective in a given instance is not predictable.
In addition, with the advent of gene therapy it has been reported that some success has been accomplished with using genes or "naked DNA" as vaccines. However, as with some of the conventional vaccines, the immune response obtained was insufficient to afford immunization. Accordingly, there is a need for additional effective adjuvant preparations which are suitable for potentiating vaccines for animals in general, and particularly in humans.
Summary of the Invention
The present invention relates to a composition for enhancing the immune response of an animal to an infectious disease vaccine wherein the composition comprises prolactin. Preferably, the composition is human prolactin and the animal to be vaccinated is, as well, human.
The present invention further relates to a composition for enhancing the immune response of an animal to an infectious disease vaccine wherein the composition comprises prolactin cDNA. Human prolactin cDNA is preferred. In another aspect, the invention relates to a method of enhancing the immune response of a subject animal to an infectious disease vaccine comprising co-administering an effective amount of prolactin or prolactin cDNA along with a vaccine.
Brief Description of the Drawing
Figure 1 shows the amino acid sequence for the prolactin protein.
Figure 2 shows the nucleic acid sequence for the prolactin cDNA.
Figure 3 is a graph illustrating the Bovine serum albumin (BSA)-specific antibody response of rats immunized with BSA alone or BSA + prolactin.
Figure 4 is a graph illustrating a comparison of the BSA- specific proliferative response of rat PBL, at 101 day time point, between four rats receiving BSA alone versus BSA + prolactin. Detailed Description of the Invention
Definitions
As used herein, "prolactin" refers to a polypeptide obtained from tissue cultures or by recombinant techniques and other techniques known to those of skill in the art, exhibiting the spectrum of activities characterizing this protein. The word includes not only human prolactin (hPRL), but also other mammalian prolactin such as, e.g., mouse, rat, rabbit, primate, pig and bovine prolactin. The amino acid sequence of a recombinant hPRL is shown in Figure 1. The recombinant PRL (r-PRL) is preferred herein.
The term "recombinant prolactin", designated as r-PRL, preferably human prolactin, refers to prolactin having comparable biological activity to native prolactin prepared by recombinant DNA techniques known by those of skill in the art. In general, the gene coding for prolactin is excised from its native plasmid and inserted into a cloning vector to be cloned and then inserted into an expression vector, which is used to transform a host organism. The host organism expresses the foreign gene to produce prolactin under expression conditions.
As used herein, the term "adjuvant" has its conventional meaning, i.e., the ability to enhance the immune response to a particular antigen. Such ability is manifested by a significant increase in immune-mediated protection. Furthermore, the term "genetic adjuvant" refers to prolactin cDNA which comprises the complement to the DNA sequence encoding the prolactin protein as defined above. The sequence for prolactin cDNA is shown in Figure 2.
General Method
Formulations containing prolactin for adjuvant purposes are most conveniently administered by intramuscular or subcutaneous injections or intraperitoneal although other methods of administration are possible. Standard formulations are either liquid injectables or solids which can be taken up in suitable liquids as suspensions or solutions for injection. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and so forth. Nontoxic auxiliary substances, such as wetting agents, buffers, or emulsifiers may also be added.
Sustained and continuous release formulations are of considerable variety and could be used in the method of the present invention, as is understood by those skilled in the art. Prolactin can be administered separately from the vaccine or in combination with the vaccine. When prolactin is combined with the vaccine, the composition administered contains an immunogen that is effective in eliciting a specific response to a given pathogen or antigen, a pharmaceutically acceptable vaccine carrier and an immunopotentiating amount of prolactin. The vaccine will normally be administered per manufacturer's instructions. Other adjuvants may be administered either with the vaccine or together with the prolactin. Prolactin will typically be used to enhance the protection afforded by animal or human vaccines that are considered "weak" (i.e., provide diminished protection in terms of level, extent, and/or duration). Examples of such vaccines are bacterins such as Pseudomonas Staphylococcal, Enterotoxin Streptococci, cytomegalovirus, HIV, Bordetella bacterin, Escherichia coli bacterins, Haemophilus bacterins, Leptospirosis vaccines, Moraxella bovis bacterin, Pasteurella bacterin and Vibrio fetus bacterin and attenuated live or killed virus products such as bovine respiratory disease vaccine (infectious bovine rhinotracheitis, parainfluenza-3, respiratory syncytial virus), bovine virus diarrhea vaccine, equine influenza vaccine, feline leukemia vaccine, feline respiratory disease vaccine (rhinotracheitis-calicipneumonitis viruses), canine parvovirus vaccine, transmissible gastroenteritis vaccine, pseudorabies vaccine, and rabies vaccine. In addition, because we have demonstrated in vitro and in vivo data that indicate that prolactin can enhance the immune response to an immunogen and thereby function as a vaccine adjuvant, it is believed that the exogenous administration of the prolactin gene would result in the expression of prolactin in vivo which would be available to function as an adjuvant to any immunogen whether administered through conventional means or via gene inoculation. The "genetic adjuvant" could be produced by inserting prolactin cDNA into a DNA delivery vehicle (e.g., plasmid vectors, liposomes, viral vectors). This could be accomplished as described by Pellegrini I., et al., Molec. Endocrinolgy, 6, 1023 (1992), Maniatis T., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press (1989) and Felger P., et al., Proc. Natl. Acad. Sci., 84, 7413, (1991 ). The "genetic adjuvant" is then administered along with either cDNA encoding the immunogen in an appropriate delivery vehicle or "naked" (i.e., solely the cDNA). In addition, the "genetic adjuvant" could be administered along with the immunogen itself. The injection sequence would be optimized per immunogen, i.e., the prolactin cDNA could be co-administered with the immunogen or immunogen cDNA, or administered in advance or subsequent to their administration. It is believed that the prolactin cDNA could be inserted into the same DNA delivery vehicle. Various routes of administration could be used.
EXAMPLE 1
Co-mitogenicity of recombinant human prolactin (r-hPRL)
Peripheral blood lymphocytes (PBL) were isolated from the blood of normal human volunteers by density gradient centrifugation on Ficoll Paque (Pharmacia). Heparinized blood was diluted 3 fold in phosphate-buffered saline (PBS) and centrifuged at 2000 rpm for 20 minutes. The buffy coat, located on the surface of the red blood cell pellet and consisting of white blood cells, was collected and diluted with an equal volume of PBS. The diluted buffy coat was layered on Ficoll Paque (6 mis of buffy coat on 4 mis of Ficoll Paque in a 15 ml tube) and centrifuged for 30 minutes at 1400 rpm. The PBL layer, found at the Ficoll-plasma interface, was collected and the cells were washed three times in PBS. PBL were then resuspended at 2x106/ml in serum-free AIM-V medium from Gibco and added to the wells of round bottom 96 well microtiter plates in a 100 μl volume (2x105 PBL/well).
A suboptimal dose of the T cell mitogen concanavalin A (Con A; 0.2 μg/ml) was added in a 50 μl volume together with 50 μl of varying concentrations of r-hPRL (0-1000 ng/ml final). Cultures were done in triplicate. The cells were incubated at 37°C/5% CO2 for 72 hours and the amount of proliferation measured by tritiated thymidine incorporation.
Tritiated thymidine (0.5μ Ci/well) was added for the last 18 hours of incubation and cell-associated radioactivity was measured by scintillation counting after harvesting the cells onto glass fiber filters using a Skatron 96 well cell harvester. Results, obtained with cells from different individuals, shown in Table 1 below, indicated that r-hPRL was able to enhance the proliferative response of T lymphocytes to a suboptimal concentration of Con A. This co-mitogenic activity was best observed with r-hPRL concentrations of 1 -10 ng/ml, illustrated in Figure 3.
Table 1
Co-mitoαenic activity of recombinant human prolactin (cpm +/- SEM)
Con A + r-hPRL (no/ml) Donor 1 Donor 2 Donor 3 No prolactin 22323±4585 35942±81 0 1 6549±1 61 8
0.1 22949±2003 34040±1446 1708311895
1 35882 3665 45839±2137 2759013151
10 32832±1972 37658±150 2299112358
100 25963±4855 35009+2105 2267411662
1000 23990±1534 3592111690 2664612574 EXAMPLE 2
Enhancement of antigen-specific proliferation bv r-hPRL
To test the ability of r-hPRL to enhance the proliferative response of human T cells to a specific antigen, PBL were incubated with various concentrations of r-hPRL and streptokinase, a common antigen to which most individuals are exposed. Cultures were performed in triplicate in the wells of 96 well round bottom microtiter plates and consisted of 100 μl PBL (2x105/well), 50 μl streptokinase (25 μg/ml final) and 50 μl of r-hPRL at varying concentrations (0-1000 ng/ml final). Proliferation was measured by tritiated thymidine incorporation after 6 days of culture at 37°C/5%C02-
The results, shown in Table 2 below, indicated that r- hPRL, at a concentration of 1 ng/ml, significantly enhanced streptokinase-induced proliferation.
Table 2
Effect of recombinant human prolactin on streptokinase-specific proliferation
Streptokinase + r-hPRL (ng/mh Proliferation (cpm +/- SEM)
No prolactin 3180714235
0.1 3022015448
1 5096416469
10 35620111318
100 3671312230
1000 3349417990 EXAMPLE 3
Effect of prolactin in enhancing the immune response to an immunogen
Twenty-four 150 gram male Sprague-Dawley rats were divided into 4 groups. The control group received an intraperitoneal injection of 10 μg BSA mixed with alum. The other 3 groups received intraperitoneal injections of 10 μg BSA mixed with alum along with either 180 μg prolactin, 375 μg prolactin or 750 μg prolactin. Tail vein bleeds were taken weekly for 4 weeks and the serum evaluated for antibody to BSA by a Radioimmunosorbent Assay (RIA). The animals were boosted after the 4th bleed with 10μg BSA mixed with alum. Tail vein bleeds were taken over a 7 week period to obtain serum which was evaluated for the development of antibody to BSA by RIA.
Bovine serum albumin (BSA)-specific proliferation of peripheral blood lymphocytes from rats immunized with BSA +/- r-hPRL To measure the effect of r-hPRL on the cellular response of rats immunized with BSA, blood was collected from individual animals sacrificed 101 days after boosting. To isolate peripheral blood lymphocytes (PBL), blood samples were diluted 4 fold in the phosphate-buffered saline (PBS) and centrifuged at 2000 rpm for 20 minutes. The buffy coat was collected and contaminating red blood cells were removed by the addition of Tris-ammonium chloride lysis buffer followed by a 10 minute incubation at 37°C. PBL were then washed twice in PBS and resuspended at 5x106/ml in RPMI-1640 medium supplemented with 100 u/ml penicillin, 100 μg/ml streptomycin, 20 mM Hepes buffer, 2 mM L-glutamine, 5x10" 5 M 2-mercaptoethanol and 5% heat-inactivated fetal calf serum. PBL were added to the wells of flat bottom 96 well microtiter plates in a 100 μl volume (5x10^ cells/well) and cultured in the presence of medium alone (background control) or 1000 μg/ml BSA added in a 100 μl volume. Cultures were done in triplicate. Proliferation was measured by tritiated thymidine incorporation after 5 days of culture at 37°C/5% Cθ2-
The results indicated that, overall, PBL rats immunized with BSA + 180 μg rhPRL displayed higher levels of BSA- specific proliferation than PBL from rats immunized with antigen alone. This observation suggests that r-hPRL may act to enhance the cellular component of the immune response to an immunizing antigen. Results are compiled in Table 3 below and are illustrated in Figures 5 and 6.
Table 3 BSA-specific proliferation of rat PBL (cpm +/- SEM)
101 days after boosting Group Background BSA-specific response
BSA alone
Rat 1 918 1 35 1236 1 100
Rat 2 559 1 169 1392 1 185
Rat 3 614 1 51 930 1 265
Rat 4 242 1 21 2122 1 257
BSA + 180 μα PRL
Rat 1 426 1 99 2552 1 30
Rat 2 269 1 18 756 1 37
Rat 3 723 1 185 4328 1 77
Rat 4 676 1 29 2023 1 397
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Richards, Susan
Kaplan, Johanne Moscicki, Richard
(ii) TITLE OF INVENTION: PROLACTIN AS ADJUVANT
(iii) NUMBER OF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: William G. Gosz (B) STREET: One Kendall Square
(C) CITY: Cambridge
(D) STATE: MA
(E) COUNTRY: U.S.A. (F) ZIP: 02139
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Gosz, William G
(B) REGISTRATION NUMBER: 27,787 (C) REFERENCE/DOCKET NUMBER: GEN 4-2.0 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 5088722583
(B) TELEFAX: 6173747225
(2) INFORMATION FOR SEQ ID NO:1 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 351 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: human prolactin
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 :
Thr He Gly Phe His Met Pro Arg Leu Cys His Glu Cys Lys Phe Arg 1 5 10 15
Met Thr Thr Arg Ala Asn Ser Leu Ala Thr Glu Phe His Met Pro Arg 20 25 30
Leu Ser Glu Gin Cys His Glu Cys Lys Phe Arg Met Thr Gly Glu Asn 35 40 45
Glu Arg Ala Thr Glu Asp Ser Tyr Met Asx Leu Ser Thr His Met Pro
50 55 60 Arg Leu Leu Cys Ser His Met Pro Arg Leu Asx Pro Met Arg Asn Ala 65 70 75 80
Glu Asn Thr Glu Arg Glu Asp Asp Glu Phe He Asn lie Thr He Asn
85 90 95
His Met Ala Asn Pro Arg Glu Pro Arg Leu Ala Cys Thr He Asn Pro 100 105 110
Arg Leu Met Arg Asn Ala Ala Cys Cys Glu Ser Ser He Asn His Met 115 120 125
Pro Arg Leu Pro Glu Pro Leu Glu Asn Gly Thr His Leu Tyr Cys His 130 135 140
Glu Cys Lys His Met Pro Arg Leu Leu Pro He Cys Pro Gly Gly Ala 145 150 155 160
Ala Arg Cys Gin Val Thr Leu Arg Asp Leu Phe Asp Arg Ala Val Val
165 170 175
Leu Ser His Tyr He His Asn Leu Ser Ser Glu Met Phe Ser Glu Phe 180 185 190
Asp Lys Arg Tyr Thr His Gly Arg Gly Phe He Thr Lys Ala He Asn 195 200 205
Ser Cys His Thr Ser Ser Leu Ala Thr Pro Glu Asp Lys Glu Gin Ala 210 215 220
Gin Gin Met Asn Gin Lys Asp Phe Leu Ser Leu He Val Ser He Leu 225 230 235 240
Arg Ser Trp Asn Glu Pro Leu Tyr His Leu Val Thr Glu Val Arg Gly
245 250 255 Met Gin Glu Ala Pro Glu Ala He Leu Ser Lys Ala Val Glu He Glu 260 265 270
Glu Gin Thr Lys Arg Leu Leu Glu Gly Met Glu Leu He Val Ser Gin 275 280 285
Val His Pro Glu Thr Lys Glu Asn Glu He Tyr Pro Val Trp Ser Gly 290 295 300
Leu Pro Ser Leu Gin Met Ala Asp Glu Glu Ser Arg Leu Ser Ala Tyr 305 310 315 320
Tyr Asn Leu Leu His Cys Leu Arg Arg Asp Ser His Lys He Asp Asn 325 330 335
Tyr Leu Lys Leu Leu Lys Cys Arg He He His Asn Asn Asn Cys 340 345 350
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1100 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
TGCCTCATTA ACTAACCACT CACATTAAAA GAAATATAAC ATATATATTA AAAATAATCA 60
TATCCTATAA TAATTAACTC ATCTAAAATA CAACCTACTG TACCATATAC TAACTGAATA 120
AGACTAGCAT TATTATTCAG GATAACTAAG TCCATAAGAT ATGTACCATA TTATACACAT 180
TTATAGCACG GATATTACTT ACTGGATATA CTTTGATCTA TCTTGATATT TATTATTCAA 240
AATACTACGT GATATATCGC ATGTCCCAAA CATGAACATC AAAGGATCGC CATGGAAAGG 300
GTCCCTCCTG CTGCTGCTGG TGTCAAACCT GCTGCTGTGC CAGAGCGTGG CCCCCTTGCC 360
CATCTGTCCC GGCGGGGCTG CCCGATGCCA GGTGACCCTT CGAGACCTGT πGACCGCGC 420
CGTCGTCCTG TCCCACTACA TCCATAACCT CTCCTCAGAA ATGTTCAGCG AATTCGATAA 480
ACGGTATACC CATGGCCGGG GGTTCATTAC CAAGGCCATC AACAGCTGCC ACACTTCTTC 540
CCTTGCCACC CCCGAAGACA AGGAGCAAGC CCAACAGATG AATCAAAAAG ACTTTCTGAG 600
CCTGATAGTC AGCATATTGC GATCCTGGAA TGAGCCTCTG TATCATCTGG TCACGGAAGT 660
ACGTGGTATG CAAGAAGCCC CGGAGGCTAT CCTATCCAAA GCTGTAGAGA TTGAGGAGCA 720
AACCAAACGG CTTCTAGAGG GCATGGAGCT GATAGTCAGC CAGGTTCATC CTGAAACCAA 780
AGAAAATGAG ATCTACCCTG TCTGGTCGGG ACTTCCATCC CTGCAGATGG CTGATGAAGA 840
GTCTCGCCTT TCTGCTTATT ATAACCTGCT CCACTGCCTA CGCAGGGATT CACATAAAAT 900
CGACAATTAT CTCAAGCTCC TGAAGTGCCG AATCATCCAC AACAACAACT GCTAAGCCCA 960
CATCCATTTC ATCTATTTCT GAGAAGGTCC TTAATGATCC GTTCCATTGC AAGCTTCTTT 1020 TAGTTGTATC TCTTTTGAAT CCATGCTTGG GTGTAACAGG TCTCCTCTTA AAAAATAAAA 1080
ACTGACTCGT TAGAGACATC 1100

Claims

Claims
1 . A composition for enhancing the immune response of an animal to an infectious disease vaccine, wherein the composition comprises prolactin.
2. The composition of claim 1 wherein the prolactin is human prolactin.
3. A composition for enhancing the immune response in accordance with claim 1 wherein the animal is a human.
4. The composition of claim 1 wherein the prolactin comprises an amino acid sequence selected from all or a portion of the amino acid sequence of Figure 1 (SEQ ID
NO.:1).
5. A composition for enhancing the immune response of an animal to an infectious disease vaccine wherein the composition comprises prolactin cDNA.
6. The composition of claim 5 wherein the prolactin is human prolactin cDNA.
7. A composition for enhancing the immune response in accordance with claim 5 wherein the animal is a human.
8. The composition of claim 5 wherein the cDNA comprises a cDNA sequence selected from all or a portion of the cDNA sequence of Figure 2 (SEQ ID NO.:2).
9. A method of enhancing the immune response of a subject animal to an infectious disease vaccine comprising co- administering an effective amount of prolactin along with a vaccine.
10. The method of claim 9 wherein the prolactin is human prolactin.
1 1 . A method for enhancing the immune response in accordance with claim 9 wherein the animal is a human.
12. The method of claim 9 wherein the prolactin comprises an amino acid sequence selected from all or a portion of the amino acid sequence of Figure 1 (SEQ ID NO.:1 ).
13. A method of enhancing the immune response of a subject animal to an infectious disease vaccine comprising co- administering an effective amount of prolactin cDNA along with a vaccine.
14. The method of claim 13 wherein the prolactin is human prolactin.
15. A method for enhancing the immune response in accordance with claim 13 wherein the animal is a human.
16. The method of claim 13 wherein the prolactin comprises an cDNA sequence selected from all or a portion of the cDNA sequence of Figure 2 (SEQ ID NO.:2).
EP95910999A 1994-02-14 1995-02-14 Prolactin as a vaccine adjuvant Withdrawn EP0952846A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US19635094A 1994-02-14 1994-02-14
US196350 1994-02-14
PCT/US1995/001866 WO1995021625A1 (en) 1994-02-14 1995-02-14 Prolactin as a vaccine adjuvant

Publications (2)

Publication Number Publication Date
EP0952846A1 true EP0952846A1 (en) 1999-11-03
EP0952846A4 EP0952846A4 (en) 1999-11-03

Family

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EP95910999A Withdrawn EP0952846A1 (en) 1994-02-14 1995-02-14 Prolactin as a vaccine adjuvant

Country Status (5)

Country Link
EP (1) EP0952846A1 (en)
JP (1) JPH09509415A (en)
AU (1) AU700104B2 (en)
CA (1) CA2183260A1 (en)
WO (1) WO1995021625A1 (en)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2232682A1 (en) * 1996-04-30 1997-11-06 Susan M. Richards Use of prolactin as a tgf-beta antagonist
EP1049485A1 (en) * 1997-01-21 2000-11-08 Genzyme Corporation Enhancement of hematopoietic cells
DE69933707T2 (en) 1999-08-25 2007-08-23 Accuplex, L.L.C. SERUM AMYLOID A AS A MARKER FOR INFLAMMATORY RESPONSE, MILK QUALITY AND THE PRESENCE OF KOLOSTRUM IN MILK
CA2387907A1 (en) 1999-10-22 2001-05-03 The Board Of Regents Of The University Of Nebraska Serum amyloid a isoform from colostrum
US20050107315A1 (en) * 2001-11-21 2005-05-19 Board Of Regents Of The University Of Nebraska Mammary-associated serum amuloid a3 promoter sequences and used for same
US7368546B2 (en) 2003-01-21 2008-05-06 The Board Of Regents Of The University Of Nebraska Human SAA3 nucleic acid molecule, protein, and methods of use for same

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990002559A1 (en) * 1988-09-15 1990-03-22 Public Health Laboratory Service Board Pharmaceutical compositions for eliciting an immunostimulant effect

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4725549A (en) * 1980-09-22 1988-02-16 The Regents Of The University Of California Human and rat prolactin and preprolactin cloned genes
US5028591A (en) * 1987-09-14 1991-07-02 Pitman-Moore, Inc. Method for stimulating the immune system

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990002559A1 (en) * 1988-09-15 1990-03-22 Public Health Laboratory Service Board Pharmaceutical compositions for eliciting an immunostimulant effect

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO9521625A1 *

Also Published As

Publication number Publication date
AU1876595A (en) 1995-08-29
JPH09509415A (en) 1997-09-22
AU700104B2 (en) 1998-12-24
CA2183260A1 (en) 1995-08-17
WO1995021625A1 (en) 1995-08-17
EP0952846A4 (en) 1999-11-03

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